Autoantibody inhibitors

ABSTRACT

The invention provides methods and compositions for inhibiting pathogenic binding of an pathogenic autoantibody to a myelin oligodendrocyte glycoprotein (MOG) autoantigen and screening for inhibitors of pathogenic binding of an autoantibody to a MOG autoantigen.

RELATED APPLICATIONS

[0001] This application claims priority under 35USC120 to U.S. Ser. No.09/384,036, filed Aug. 26, 1999, which claims the benefit of U.S.Provisional Application 60/097,953 filed Aug. 26, 1998, both of whichare incorporated herein by reference.

[0002] This invention was made with Government support under Grant No.AI43073, awarded by the National Insitutes of Health. The Government hascertain rights in this invention.

INTRODUCTION

[0003] 1. Field of the Invention

[0004] The field of this invention is polypeptide autoantibodyinhibitors and methods of use thereof.

[0005] 2. Background

[0006] Multiple sclerosis (MS) is a chronic relapsing remitting disorderdisease of the central nervous system that affects 350,000 Americansand, second to trauma, is the leading cause of disability among youngadults. MS is an immune-mediated disorder characterized pathologicallyby perivenular white matter infiltrates comprised of macrophages andmononuclear cells (inflammation), and destruction of the myelin sheathsthat insulate nerve fibers (demyelination).

[0007] Experimental allergic encephalomyelitis (EAE) in rodents has beenthe most widely employed model for testing of therapies for human MS.These traditional disease models for MS generally have promoted theconcept that MS is a T-cell-mediated disorder. However, the autoantigensthat serve as targets for the immune attack have not been identified andthe molecular mechanisms implicated in myelin damage remain uncertain.While it is clear that CNS inflammation in EAE is initiated byautoagressive T-cells that recognize myelin antigens in the context ofclass II-MHC molecules, many of the models lack the early demyelinatingcomponent of the MS lesion. B-cell activation and antibody responsesappear necessary for the full development of EAE and earlier studies onimmune mediated demyelination using myelinated cultures of CNS tissuehave implicated humoral factors as effector mechanisms. Thus, it is notsurprising that rodent EAE has not been a robust predictor of efficacyin humans as fundamental differences in the clinical course, pathology,and immunologic response to myelin proteins distinguish rodent EAE fromhuman MS.

[0008] Recently a novel MS-like illness in an outbred nonhuman primate,the common marmoset Callithrix jacchus, has been defined. The marmosetEAE has a prominent, MS-like early demyelinating component whichrequires the presence of myelin-specific autoantibodies, and hasafforded an opportunity to understand the interactions between theseantibodies and their target antigens on myelin. Characteristics of themodel include: a. Mild clinical signs and a relapsing remitting coursesimilar to MS; b. A primary demyelinating pathology with early gliosisindistinguishable from MS lesions (demyelinating plaques); c. Naturalbone marrow chimerism permitting successful adoptive transfer ofencephalitogenic (e.g. disease-inducing) T-cell clones and lines; d.Diversity of the encephalitogenic repertoire of T-cells reactive againstthe major myelin protein myelin basic protein (MBP); e. Differentdisease phenotypes resulting from immunization with different myelinconstituents: in contrast to whole myelin, immunization with MBPproduces a non-demyelinating form of EAE; f. Demonstration thatdemyelination is antibody-mediated but also requires an encephalitogenicT-cell response to facilitate autoantibody access to the nervous system;and, g. A key role of myelin oligodendrocyte glycoprotein (MOG) inplaque formation: adoptive transfer of anti-MOG antibody innon-demyelinating MBP-EAE reproduces fully developed MS-like pathology.

[0009] The highly immunogenic properties of MOG (<0.05% of total myelinprotein) may be related to its extracellular location on the outermostlamellae of the myelin sheath, where it is accessible to pathogenicantibody in the context of blood brain barrier disruption byencephalitogenic T-cells. The C. jacchus model permits preciseidentification of cellular and humoral immune responses that result inan MS-like lesion in a species with immune and nervous system genes thatare 90-95% homologous to humans. The relevance of this model to human MSis emphasized by the recent finding of strong T-cell and antibodyresponses to MOG in MS patients.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to autoantibody inhibitors andmethods of use thereof. Accordingly, the invention provides methods andcompositions for inhibiting pathogenic binding of an autoantibody to anautoantigen and screening for inhibitors of pathogenic binding of anautoantibody to an autoantigen.

[0011] In one aspect, the present invention provides a compositioncomprising a peptide consisting of residues 28-36, 13-21, 62-74, 27-34or 40-45 of rat, human or marmoset MOG. In a preferred embodiment theMOG polypeptide is directly joined at its N- and C- termini with otherthan natural human or marmoset MOG flanking residues.

[0012] In another aspect, the present invention provides a method ofinhibiting pathogenic binding of a MOG specific autoantibody to MOG oran immunodominant epitope thereof.

[0013] In yet another aspect, the present invention provides a method ofdetecting autoantibodies in a tissue sample. In a preferred embodiment amethod of identifying autoantibodies against myelin/oligodendrocyteglycoprotein (MOG) within lesions of human MS and C. jacchus EAE, wherethey appear to be directly responsible for the disintegration of themyelin sheaths, is provided.

[0014] In a further aspect, the present invention provides a method ofscreening small molecules or candidate agents capable of binding to anautoantigen and thereby inhibit binding of an autoantibody. The methodcomprises contacting a solution comprising an autoantigen and anautoantibody, incubating under conditions sufficient to allow thereaction to reach equilibrium, and comparing the binding of theautoantibody in the absence of the small molecule inhibitor or candidateagent to the binding of the autoantibody in the presence of the smallmolecule inhibitor or candidate agent. In a preferred embodiment thesmall molecules specifically bind at least one immunodominant epitope ofthe autoantigen.

[0015] In yet another aspect of the invention there is provided a methodof inhibiting pathogenic binding of an autoantibody to an autoantigencomprising administering to a host subject to pathogenicautoantigen-autoantibody binding-mediated pathology an effective amountof a composition comprising a fragment of an antibody specific for theautoantigen sufficient to specifically bind the autoantigen andcompetitively inhibit the binding of an autoantigen-specificautoantibody to the autoantigen, wherein the fragment does not comprisea functional Fc portion of the autoantigen-specific antibody. In apreferred embodiment, the autoantigen-autoantibody binding is associatedwith a demyelinating disease of the central or peripheral nervoussystem. In a particular embodiment, the disease is associated withpathogenic autoantibody binding, such as MS, lupus, arthritis ordiabetes. In more particular embodiments, the autoantigen is a MOGautoantigen and the fragment is a F(ab′)₂ fragment.

[0016] In yet another aspect, the invention also provides methods ofscreening for an agent which modulates the binding of an autoantibody toan autoantigen. Such methods generally involve incubating a mixturecomprising the autoantibody or an auto antibody-specific bindingfragment thereof, the autoantigen, and a candidate agent underconditions whereby, but for the presence of said agent, the autoantibodyor fragment thereof specifically binds the autoantigen at a referenceaffinity; detecting the binding affinity of autoantibody or fragmentthereof to the autoantigen to determine an agent-biased affinity,wherein a difference between the agent-biased affinity and the referenceaffinity indicates that said agent modulates the binding of theautoantibody or fragment thereof to the autoantigen. In particularembodiments, the autoantibody or fragment thereof is a F(ab′)₂ fragment;the autoantigen comprises a MOG epitope; and/or the autoantigencomprises a MOG epitope consisting of residues 28-36, 13-21, 62-74,27-34 or 40-45 of rat, human or marmoset MOG.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The following description and examples are offered by way ofillustration and not by way of limitation.

[0018] The invention provides methods and compositions for inhibitingpathology associated with the binding of an autoantibody to a MOGpolypeptide, such as occurs in MS. The general methods comprise the stepof administering to a host, subject to a pathogenic MOGpolypeptide-autoantibody binding, an effective amount of a compositioncomprising a MOG polypeptide-specific antibody fragment not having afunctional Fc portion and sufficient to specifically bind the MOGpolypeptide and competitively inhibit the binding of the autoantibody tothe MOG polypeptide, whereby the pathology is inhibited. In a particularembodiment, the fragment is selected from the group consisting of Fv,F(ab′)₂, F(ab), F(ab)₂ or fragments thereof.

[0019] The compositions include pharmaceutical compositions comprising aMOG polypeptide-specific antibody fragment sufficient to specificallybind a natural MOG polypeptide and competitively inhibit the binding ofan autoantibody to the MOG polypeptide, wherein the fragment does notcomprise a functional Fc portion, and a pharmaceutically acceptablecarrier. The compositions may also comprise a MOG tolerogenic T-cellepitope which induces tolerance and acts synergistically with theantibody fragment to inhibit pathology.

[0020] In another embodiment, the invention provides methods andcompositions for detecting the presence of an autoantibody bound to afirst autoantigen in a tissue. These methods generally comprise thesteps of contacting the tissue with a second, labeled autoantigen underconditions wherein the autoantibody binds the second autoantigen to formfirst autoantigen-autoantibody-second autoantigen labeled complexes, andspecifically detecting the labeled complexes. The first and secondautoantigens are generally the same or at least include epitopes of thesame autoantigen. Preferred autoantigens include, but are not limited tomyelin oligodendrocyte glycoprotein (MOG), myelin associatedglycoprotein (MAG), myelin/oligodendrocyte basic protein (MOBP),Oligodendrocyte specific protein (Osp), myelin basic protein (MBP),proteolipid apoprotein (PLP), galactose cerebroside (GalC), glycolipids,sphingolipids, phospholipids, gangliosides and other neuronal antigens.

[0021] In yet another embodiment, the invention provides methods andcompositions for detecting MOG polypeptide-specific B-cells. Suchmethods generally comprise the steps of fractionating blood to obtain anunselected population of B-cells comprising rare MOGpolypeptide-specific B-cells, contacting the population with labeled MOGpolypeptides under conditions whereby the labeled MOG polypeptides bindsthe rare MOG polypeptide-specific B-cells to form labeled complexes ofthe labeled MOG polypeptides and the rare MOG polypeptide-specificB-cells, and specifically detecting the complexes.

[0022] In yet another embodiment, the invention provides methods andcompositions for screening for a candidate agent to inhibit pathologyassociated with MOG polypeptide-specific antibody binding to a MOGpolypeptide. These methods generally comprise the steps of:

[0023] incubating a mixture comprising: the antibody or a MOG-specificfragment thereof, the MOG polypeptide, and a candidate agent,

[0024] under conditions whereby, but for the presence of said agent, theantibody or fragment thereof specifically binds the MOG polypeptide at areference affinity;

[0025] detecting the binding affinity of antibody or fragment thereof tothe MOG polypeptide to determine an agent-biased affinity,

[0026] wherein a diminution of the agent-biased affinity with respect tothe reference affinity indicates that said agent inhibits the binding ofthe antibody or fragment thereof to the MOG polypeptide and provides acandidate agent for inhibiting pathology associated with MOGpolypeptide-specific antibody binding to a MOG polypeptide.

[0027] In yet another embodiment, the invention provides polypeptidescomprising MOG-specific B- and T-cell epitopes, including polypeptidescomprising a fragment having N and C ends and consisting of residues28-36, 13-21, 67-73, 27-34 or 40-45 of human, rat or marmoset MOG,wherein the fragment is directly joined at at least one of the N andC-ends with other than natural human or marmoset MOG flanking residues.Such polypeptides are useful, for example in methods of inhibiting MOGpolypeptide-autoantibody binding, such as the general method comprisingthe step of contacting a mixture of a MOG and an antibody with apolypeptide, whereby the MOG-antibody binding is inhibited.

[0028] As used herein, the term “antibody” refers to recombined immuneproteins such as T-cell antigen receptors and immunoglobulins, as wellas chimeric, humanized or other recombinant antibodies. As used herein,the term “antibody fragment” refers to fragments of antibodies such asFab, Fab′, F(ab)₂, F(ab′)₂ and Fv or any combination thereof. Fv andfragments thereof may be monovalent or divalent. Fv is also known in theart as a minimal antibody fragment. Methods of making antibodyfragments, particularly F(ab′) are known in the art. (See for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1988), incorporated herein by reference). Forexample, F(ab), Fv, etc. can also be produced by recombinant technology.

[0029] As used herein, “other than natural human or marmoset MOGflanking residues” refers to anything other than residues naturallyflanking the recited peptides in the native proteins. For example, otherthan natural flanking residues includes no flanking residues or flankingresidues different from what naturally flanks the recited peptide.

[0030] MOG was originally identified by the mouse monoclonal antibody8.18.C5, raised against rat cerebellar glycoproteins. It is aquantitatively minor protein representing only 0.01 to 0.05% of thetotal myelin proteins and has no known function within the CNS. MOG is amember of the immunoglobulin (Ig) superfamily, with animmunoglobulin-like, extracellular domain comprised of 121 amino acidscontaining one glycosylation site (Asn in position 31) and two highlyhydrophobic regions that could represent transmembrane domains, for atotal length of 224 amino acids. MOG is widely expressed onoligodendrocyte cell bodies and processes, especially on the outermostlayers of the myelin sheaths, and may be more readily accessible toantibody attack than intra-cytoplasmic MBP, or intra andinter-membranous proteolipid apoprotein (PLP). In all species studiedincluding C. jacchus, the non-glycosylated, recombinant extracellulardomain of MOG (rMOG) which is highly conserved, suffices for sensitizinganimals for EAE. In one aspect of the invention, we have identifiedminimal T-cell and B-cell epitopes, including residues 28-36, 13-21,62-74, 27-34 or 40-45; natural human and rat MOG sequences are known inthe art; natural marmoset MOG is identical to the human except for thefollowing substitutions: 9S, 13Q, 19A, 20A, 42S, 60E, 75D, 84K, 91P,112Q, 137F, 148Y and 151H.

[0031] The immune response in autoimmune diseases may possess bothcellular and humoral components. Our data indicate that the followingsequence of events leads to myelin destruction in CNS autoimmunedemyelination:

[0032] 1) Myelin vacuolation caused by soluble mediators (cytokines,antibodies, free radicals), and/or cellular cytotoxicity. A pattern ofintramyelinic edema similar to this has also been observed previously inthe CNS of rats intoxicated with tri-ethyl tin sulfate and,interestingly, these changes were reversible.

[0033] 2) Transformation of vacuolated myelin into networks of smallvesicles separated by 2-3 layers of altered myelin with a reducedperiodicity (5-6 nm). This dramatic transformation appears to beassociated with the deposition of MOG-specific IgG and to reflectantibody-mediated damage, possibly due to complement activation, orantibody-dependent cytotoxicity mediated by macrophages that areinvariably associated with vesicular myelin disruption. Conceivably, theinitial vacuolar lesion renders the myelin membranes accessible to anattack by autoantibodies.

[0034] 3) Macrophage activation leading to receptor-mediatedphagocytosis of the vesiculated myelin debris. This mechanism has beendemonstrated previously in MS and in EAE with IgG serving as a ligandbetween the myelin debris and Fc receptors in clathrin-coated pits onthe macrophage surface. This stage of lesion pathogenesis, althoughantibody-mediated, may be independent of antibody specificity.

[0035] As just outlined above, for example, in MS the inflammatorycomponent is T-cell mediated while the demyelinating component appearsto be B-cell mediated. Thus, effective treatments should address bothcomponents.

[0036] The present invention provides compositions comprising theimmunodominant epitopes of MOG. The abolition of the peripheral T-cellresponse by a tolerization protocol to the extracellular portion ofrecombinant MOG (aa 1-125) (rMOG; rMOG is comprised of residues 1-125 ofthe extracellular amino terminus of MOG extended by MRGS at the NH₂ andASES(H)₆ at the COOH termini) provided the basis for the presentinventive epitope-derived peptide compositions. Mapping of the criticalMOG epitopes (including 26-38 and 64-72) was accomplished by cloningT-cells from rMOG-immunized animals and by analyzing T-cell and antibodyresponses to short peptides of MOG in rMOG immunized marmosets.

[0037] Mapping of the antibody response to MOG in C. jacchus indicateslimited heterogeneity of epitope recognition by autoantibodies. We haveidentified regions of MOG that are targeted by demyelinating antibodiesusing linear peptides. The native, serum polyclonal antibodies inrMOG-immunized marmosets are directed against 4 discrete epitopes alongthe amino acid sequence, aa 13-21, 28-34, 40-45, 65-74 or shortersequences, most of which are conserved sequences across species. Thesepeptides differ from those identified to date as antibody epitopes inrodents (aa 35-55), however they bind to antibodies present within thenetwork of vesiculated myelin in acute lesions of human MS as shown inthe Examples below. Because most antibodies generally recognizediscontinuous epitopes on proteins, our analysis methodology providesdetailed knowledge of the structure of MOG is needed to fully define theantigenic repertoire of demyelinating antibodies in C. jacchus andhumans. Combinatorial libraries were then made in order to generateF(ab′)₂ fragments with high affinity for MOG capable of competing withpathogenic IgG and of inhibiting complement-mediated and antibodydependent cellular cytotoxicity. These F(ab′)₂ fragments were testedalone and in combination with T-cell tolerogenic peptides for theirability to prevent and treat disease in C. jacchus.

[0038] A recently identified patient with a progressive spinal corddisorder associated with an IgG monoclonal gammopathy reactive to MOGoffered a unique example of the pathophysiologic consequences of ananti-MOG antibody response in a natural experiment. The human monoclonalantibody was adoptively transferred into a C. jacchus withnon-demyelinating EAE. Following adoptive transfer the marmosetdeveloped demyelination. Transfer of human IgG in this species iswell-tolerated and the blocking ability of F(ab′)₂ fragments isdemonstrated in the adoptive transfer system. The antibody fragmentsretain their ability to recognize antigenic epitopes yet lack theability to activate complement or bind macrophages, they coat theautoantigen such that the endogenous autoantibodies are unable to bind apathological level.

[0039] In the preparation of the pharmaceutical compositions of thisinvention, a variety of vehicles and excipients and routes ofadministration may be used, as will be apparent to the skilled artisan.Representative formulation technology is taught in, inter alia,Remington: The Science and Practice of Pharmacy, 19th ed., MackPublishing Co., Easton, Pa., 1995; e.g. Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9^(th) Ed., 1996, McGraw-Hill.

EXAMPLES

[0040] The following examples are offered to illustrate this inventionand are not meant to be construed in any way as limiting in scope ofthis invention.

[0041] We show that antibodies against MOG that cause demyelination inmarmosets may be modified chemically and used as therapeutic tools tocompetitively block the binding of real-life, pathogenic antibody. Thisis achieved by enzymatic digestion, e.g., with pepsin, which cleaves theintact antibody into a large fragment (F[ab′]₂) that contains the sitesthat bind to the target antigen (MOG), and smaller fragments includingthe Fc portion, a portion of antibodies known to contain receptors forsystems such as complement and macrophages (also known to mediate manypathogenic or cytotoxic properties of antibodies). Thus, the F(ab′)₂retains the capacity to bind to MOG in the brain, but is devoid ofcapacity to Fc complement or activate macrophages and protects/masks MOGotherwise recognized by the pathogenic antibodies. In a particularexample, a pair of marmosets were first sensitized to EAE with MBP(non-demyelinating), then both given intravenous demyelinating antibody(mouse monoclonal 8.18.C5) against MOG. Simultaneously, one animal(control) received a placebo F(ab′)₂ injection, and the other receivedF(ab′)₂ fragments prepared from the same demyelinating antibody. Thecontrol animal showed aggravation of the clinical signs of EAE and theexperimental animal did not. The animals were sacrificed 3-5 days laterand histology of the brain and spinal cord obtained. The control animalhad evidence of demyelinating lesions, and the experimental animal hadlesions with inflammation (cellular infiltration) but no demyelination(no myelin destruction). This experiment shows that marmosets can beprotected from antibody mediated demyelination by MOG-specific F(ab′)₂fragments.

[0042] Complementary experiments of the above indicate that suchtherapeutic principle of F(ab′)₂ or F(ab′) fragments could be useful forhuman MS or related disorders: antibodies against MOG are intimatelyassociated with active lesions of MS where there is morphologic evidencefor the ongoing disintegration of myelin (see “Identification ofautoantibodies associated with demyelination in multiple sclerosis”,below). In this work, the epitopes of MOG that are recognized bymarmosets are also disclosed for the first time and this information isused to construct gold-conjugates as immunoprobes to identify thepresence of MOG-specific autoantibodies in both primate and humantissues.

Induction of Marmoset EAE

[0043] EAE was induced in marmosets as described by Genain et al. (1999)Nature Medicine 5, 170-175. Six marmosets were actively sensitized with50 to 100 μg of recombinant rat MOG dissolved in 100 μl ofphosphate-buffered saline and emulsified with an equal volume ofcomplete Freund's adjuvant (CFA) containing 3 mg/ml killed Mycobacteriumtuberculosis (h37Ra; DIFCO, Detroit, Mich.). The MOG/CFA emulsion wasgiven intradermally at four injection sites in the scapular and hipregions in a total volume of 0.2 ml. On the day of immunization withMOG/CFA, 1×10¹⁰ inactivated Bordetella pertussis organisms in 2.5 ml ofisotonic saline were given intravenously and the dose repeated 2 dayslater. For comparison, 4 marmosets sensitized with 200 mg of whole whitematter (WM)/CFA and B. pertussis were examined between 18 and 39 daysafter immunization. MOG-sensitized marmosets were maintained for up to93 days after immunization. Animals sensitized with either WM or MOGdisplayed signs of EAE within 21 days of immunization. The animals weresacrificed by intracardiac perfusion under deep anesthesia 18 to 93 daysafter immunization.

MS tissues from humans.

[0044] Human CNS tissues were obtained from 3 subjects with MS by biopsyor autopsy (8 weeks, 11 years and 17 years after diagnosis). Patient 1was an 18-year old Caucasian woman with a 3-month history of acutelydeveloping right hemiplegia, sensory loss, and spasticity. Computedtomographic scanning revealed WM hypodensity in the leftparieto-occipital region. A brain biopsy was performed forneuropathological evaluation. The resultant diagnosis was activelydemyelinating, inflammatory, edematous lesions of recent origin, typicalof a fulminant inflammatory demyelinating condition, consistent withacute MS.

[0045] Patient 2 was a 31 year old Caucasian female with an 8-yearhistory of chronic progressive MS characterized by numbness and weaknessof the limbs, gait disturbance, urinary incontinence, tremor, nystagmus,and blurred vision. Terminally, the patient was wheel-chairbound,developed seizures and aspiration pneumonia, and died. An autopsy wasperformed within 1.5 hours of death. Neuropathological examinationrevealed a predominance of small (3-5 mm), disseminated, recent,intensely inflamed, edematous, demyelinating lesions as well as larger,more established plaques with fibrous astrogliosis and well-demarcatededges.

[0046] Patient 3 was a 34-year old Caucasian female with a history ofrelapsing-remitting MS for 10 years after initial diagnosis at age 20.The disease entered a chronic progressive course for the last 7 years ofher life. At the time of death, the patient presented with bilateraloptic atrophy, internuclear ophthalmoplegia, spastic paraparesis, andmoderate limb ataxia. The cause of death was respiratory failure. Anautopsy was performed 4 hours after death. Neuropathology of this caserevealed intensely inflammatory, edematous, actively demyelinatinglesions of recent origin, as well as active chronically demyelinatedlesions.

Tissue Preparation for Analysis.

[0047] At the time of sampling, animals were sacrificed underpentobarbital anesthesia by intracardiac perfusion with 200 ml ofphosphate-buffered saline followed by 150 to 200 ml of cold PO₄-buffered2.5% glutaraldehyde. Two MOG-sensitized marmosets were sampled duringthe acute phase of the disease (14-16 days after immunization), 3 weretaken after the acute phase, either during relapses or remission (23, 25and 27 days after immunization), and 1 was examined after two relapsesat 93 days after immunization. The 4 whole WM-sensitized animals wereexamined at 18, 30, 30 and 39 days after immunization after acute onsetbut before relapse. From the glutaraldehyde-perfused animals, the CNSwas removed and routine neuropathology performed on formalin postfixed,paraffin-embedded material stained with hematoxylin and eosin, LusolFast Blue (for myelin), and the Bodian silver technique (for axons).

[0048] For fine structural analysis of marmoset tissues, 1-mm sliceswere taken form optic nerve, cerebral hemispheres, cerebellum,brainstem, medulla, and spinal cord at C7, T3, L2, L5, L6, and L7. Inaddition, samples were taken from spinal nerve roots and sciatic nerves.The slices of glutaraldehyde-fixed brain tissue were trimmed as flatrectangles (˜4×6 mm) and spinal cord was left as whole slices. From the3 cases of MS describe in Example 2, small pieces of biopsy tissue orslices of autopsied CNS material, 3 to 5 mm thick, were immersion-fixedfor 4 to 24 hours at 4° C., then cut into thin, 1-mm slices to 3 to 5 mmin diameter. Glutaraldehyde-fixed tissues were then postfixed inPO₄-buffered 1% OsO₄ for 1 hour on ice. Samples were dehydrated, clearedin propylene oxide, and embedded flat in epoxy resin. Thin (1 μm)sections of epoxy-embedded tissue were prepared for light microscopy(LM) and stained with toluidine blue or reacted for immunocytochemistry.For electron microscopy (EM), sections were placed on copper grids,contrasted with lead and uranium salts (lead citrate and uranylacetate), carboncoated, and scanned in a Siemens 101 or Hitachi H 600-S.

Ultrastructural patterns of demyelination are identical in C. jacchusEAE and in acute MS plaques.

[0049] CNS tissues from 6 C. jacchus marmosets with MOG-induced EAE andfrom 3 human subjects with MS, all showing acute lesions, were examinedby electron microscopy (EM). In marmoset EAE, large demyelinated plaquesup to several mm in diameter were disseminated throughout the CNS,invariably centered on venules and characterized by perivascularinflammation and a prominent margin along which many myelinated nervefibers displayed vacuolated myelin sheaths. This typical pattern ofmyelin vacuolation resulted from the enlargement of individual myelinsheaths due to interlamellar splitting and swelling, with the axondisplaced to one side surrounded by several layers of intact myelin.Micrographs showing the optic nerve from an animal with acute EAEinduced by immunization with 50 μg of recombinant rat MOG in adjuvant,sacrificed 3 days after onset of clinical signs demonstrated thepresence of large intramyelinic vacuoles at the perimeter of ademyelinated lesion, with axons surrounded by normal-appearing myelinsheaths elsewhere.

[0050] Between the lesion center and the margin was a broad zone ofdemyelination containing macrophages laden with myelin debris. The moststriking finding was the presence within the demyelinated zone of largenumbers of axons surrounded by aggregates of disrupted myelin rearrangedas an expanded network. These axons were displaced laterally as themembranous network gradually became dissociated from the axon and takenup by adjacent macrophages.

[0051] Demyelination of fibers in acute MS was structurally identical tothat seen in marmoset EAE, with the demyelinated axon lying within amembranous network of myelin. Elsewhere in the edematous parenchyma,free floating aggregates of myelin debris were common. Electronphotomicrographs of tissue taken from a subcortical white matter biopsyfrom an 18-year old female patient with an 8-week history of neurologicsigns, white matter hypodensity on MRI scan and a diagnosis of acute MSshowed myelin around axons transformed into a vesicular network similarto that described above. Fibrous astroglial processes, naked axons and areactive, ameboid microglial cell (below), were also identified. Highresolution analysis of the myelin networks in both marmoset EAE andhuman MS revealed vesicles surrounded by 2 to 3 layers of looselycompacted membranes with a reduced periodicity (5-6 nm) when compared tointact myelin in normal tissue.

MOG-specific autoantibodies are associated with mvelin vesiculation inthe C. jacchus EAE lesion.

[0052] MOG is a quantitatively minor myelin protein (less than 0.05% oftotal myelin proteins) with an immunoglobulin (Ig)-like extracellulardomain that is expressed in abundance on the outermost layer of myelinsheaths, which may render it accessible to antibody attack. Althoughautoantibodies against MOG have been shown to enhance demyelination inseveral EAE models, the detailed interactions between these antibodiesand myelin membranes has not been investigated. To identify the sites ofautoantibody binding within demyelinating lesions, we performedimmunocytochemistry on frozen and epoxy-embedded marmoset CNS tissuewith gold-labeled anti-human IgG antibody (cross-reactive with marmosetIgG) followed by silver enhancement.

[0053] For the demonstration of antigen-specific autoantibodies inmarmoset and human MS tissue in situ, a selection of myelin-related andcontrol peptides were directly coupled to immunogold and applied totissue sections. Immunogold labeling was performed on ultra-thinsections of frozen or fixed tissues. Gold conjugates were prepared of(1) three MOG peptides (amino acids [aa] 1-20, aa 21-40, and aa 41-60 ofhuman MOG) with known encephalitogenic activity in marmosets; (2) oneMOG peptide that has been shown not to be encephalitogenic in marmosets(aa 101-120); (3) one human myelin basic protein (MBP) peptide (aa82-101) that is encephalitogenic in marmosets and immunodominant inhumans with the DR2 haplotype; and, as a control, (4) a peptide of mouseserum albumin (MSA; aa 560-574). Peptides having human MOG subsequenceswere synthesized using standard Fmoc chemistry and purified (>95%) byHPLC (Research Genetics Inc., Huntsville, Ala.): MOG 1-20, MOG 21-40,MOG 61-80, MBP 82-101, and MSA 560-574. The gold conjugates weresynthesized by using monosulfo-N-hydroxy succinimide-Nanogold labelingreagent (particle diameter, 1.4 nm), according to the manufacturer'sinstructions (Nanoprobes, Stonybrook, N.Y.), followed by extensivedialysis to remove unreacted peptide. Immunoreactivity was detected on1-μm epoxy sections of marmoset spinal cord tissue and active MSlesions. For this, sections were etched with sodium ethoxide,equilibrated in PO₄-buffered saline containing 0.05% Triton X-100, andblocked with 10% normal rabbit serum. Sections were incubated withpeptide-immunogold conjugates (1:100 in buffer) for 2 hours at roomtemperature. After washing, detection was performed by using silverenhancement (Nanoprobes). Sections were counterstained with toluidineblue. For the detection of IgG, sections were reacted with gold-labeledanti-monkey or anti-human IgG (Nanoprobes) at 1:100.

[0054] As controls, sections were either pretreated with unlabeledencephalitogenic MOG peptides or MBP to block the reaction, reacted withunlabeled nonencephalitogenic MOG peptide (aa 101-120) beforeapplication of the gold conjugates, or treated with gold-labeledirrelevant antigens (histone or MSA) or irrelevant IgG (anti-goat). Thespecificity of the labeling with the gold conjugates of encephalitogenicMOG peptides was also assessed in western blots where the MOG proteinwas first reacted with immune marmoset serum. Full details of the testand control reagents used to determine the specificity of theimmunoreactivity can be found in Genain et al., Nature Med. 5:170-175(1999).

[0055] Sections from the lumbar region of the spinal cord from theanimals were obtained. The positive reactivity (brown coloration) ofvesiculated myelin around axons (arrows), indicated the presence of IgG.Non-demyelinating axons did not stain. Positive reactivity for IgG wasspecifically found over the vesiculated networks of disrupted myelinsurrounding axons.

[0056] We next identified the target antigens bound by theseimmunoglobulins by the application of immunogold-labeled conjugates ofselected peptides of MOG and myelin basic protein (MBP). These myelinantigens were directly labeled with the gold particles on their primaryNH₂ residues and were used to detect antigen-specific autoantibody insitu. With this technique, three separate gold-conjugated peptides ofMOG were co-localized over the networks of disintegrating myelin sheathsin a pattern similar to that observed for gold-conjugated anti-IgG.

[0057] These peptides contained the amino acid sequences of MOGrecognized by demyelinating antibodies that develop in serum ofMOG-immunized marmosets (aa 1-20, aa 21-40 and aa 61-80). Thegold-conjugated MOG peptide (aa 21-40) used has a sequence conservedacross species. MOG-reactive droplets were also seen in surroundingmacrophages, indicating the presence of internalized myelin debris towhich anti-MOG antibody was bound. Positive reactivity with the labeledantigen indicated the presence of MOG-specific antibody in situ onvesicular myelin around axons and on myelin debris within theextracellular space and macrophages. Normal myelin (around the majorityof the fibers) was not stained.

[0058] In contrast, gold-labeled conjugates of a peptide containing animmunodominant epitope of human MBP (aa 82-101, conserved across primatespecies) and of a control peptide of mouse serum albumin (MSA, aa560-574), failed to label myelin membranes or macrophages. Thus, thevesiculated myelin networks are unstained (arrows) by either thegold-conjugated peptide of MBP or the gold-conjugated peptide of MSA.

[0059] These observations demonstrate in this non-human primate model ofEAE that antibodies specific to MOG are in direct contact with thedisintegrating myelin membranes and indicate that formation of thevesiculated membranous networks resulted from lytic attack by theseautoantibodies.

MOG-specific autoantibodies are associated with myelin vesiculation inlesions of acute human MS.

[0060] We next investigated with similar immunogold labeling thepresence of MOG- and MBP-specific autoantibodies in CNS tissue obtainedat biopsy or autopsy from patients with MS. As in marmoset EAE,gold-conjugated anti-IgG labeled the membranous myelin networks aroundsingle demyelinating axons, along with droplets of myelin debrisscattered throughout the parenchyma. IgG is localized along thedisintegrated myelin sheath of an axon cut in longitudinal section;cytoplasm of an hypertrophied astrocyte; tangential section of anoligodendrocyte. Densely stained IgG-coated myelin debris are visible inthe parenchyma and in 3 macrophages (probably ameboid microglia). Inaddition, occasional plasma cells showed positive staining by anti-IgG.With the immunogold-labeled myelin antigen conjugates, vesiculatedmyelin networks were intensely stained by gold-conjugated-MOG peptides,and to a lesser extent by gold-conjugated MBP but not by MSA.

[0061] IgG-myelin complexes labeled with gold-conjugates of MOG and MBPwere also present in macrophages but not in astrocytes oroligodendrocytes. No MOG- or MBP-labeled plasma cells were encountered.Reactivity with gold-conjugates was not observed in normal appearing MSwhite matter or around perivascular inflammatory cuffs. In marmosetsimmunized against whole myelin, a similar pattern of both anti-MOG andanti-MBP Ig deposition was observed. CNS tissue from amyotrophic lateralsclerosis, another neurologic disorder associated with white matterdamage and macrophage activity, failed to show myelin antigen- specificimmunogold reactivity. These findings directly identify MOG-specificantibodies in actively demyelinating lesions of human MS, indicatingthat, as in MOG-induced EAE, these autoantibodies play a causal role inthe formation of small vesicles in the disrupted myelin sheaths. Solubleand B-cell surface Ig with anti-MBP specificity have been described inMS brain tissue, and in the current study, MBP-specific Ig was localizedwithin the vesiculated myelin networks in MS lesions. Although anti-MBPantibodies have not been shown experimentally to initiate demyelinatingpathology, these autoantibodies can mediate separate pathogenicmechanisms such as receptor-mediated phagocytosis by macrophages and/orpresentation of myelin autoantigens to specific T-cells.

[0062] It is noteworthy that autoantibodies appear to be boundexclusively to the small vesicles that characterize the stage ofcomplete disintegration of the myelin membranes, and to the myelindebris present either in the extracellular space or in phagocytic cells.Interestingly, similar but less extensive vesiculation of myelin wasreported in earlier studies of rodent EAE where it was perceived as atransient early phenomenon. However, in the marmoset where lesionformation is protracted and ever expanding, the disrupted myelin wasfound consistently. The large scale vacuolation of myelin at the lesionmargin among normally myelinated fibers occurred in the absence ofsignificant local cellular infiltration or IgG deposition, and has alsobeen reported at the edge of active MS lesions. This change in themyelin structure could be mediated by soluble factors diffusing from thecenter of the demyelinating plaque or from activated glial cells at theedge of the lesion. Morphologic changes similar to these large vacuoleshave been reported in myelinated CNS cultures exposed to TNF-alpha andto a lesser degree, in cultures exposed to serum from animals with EAEand from subjects with MS.

[0063] Many of the therapeutic approaches targeting pathogenic T-cellresponses in EAE models have not yet translated into successfultreatment for human MS, perhaps suggesting that other components of theimmune system need to be taken into account. B-cell responses appear tobe a key factor for severity of clinical disease and pathology in C.jacchus EAE. The current results underscores the role of autoantibodiesin the widespread destruction of myelin in MS, and emphasizes that indiseases that are initiated by T-cell responses, antibodies againstcritical antigens of the target organ are essential for development ofirreversible tissue damage.

In vivo Administration of MOG-specific F(ab′)₂ Fragments.

[0064] Marmosets were administered 1 mg MBP in CFA containing B.Pertussis at Day 0 inducing non-demyelinating EAE. On Day 21 the animalswere administered intravenously 0.17 mmol/kg of the F(ab′)₂ from either8.18.C5, a murine monoclonal anti-MOG antibody, or an anti-Influenza-A(control) antibody, then administered 0.17 mmol/kg 8.18.C5 antibodyfollowed by a second intravenous administration of the appropriateF(ab′)₂ for two hours. The animals were euthanized on day 35. Tissuesamples were prepared as described in Example 3. Using high resolutionmicroscopy and immunogold-labeled peptides of myelin antigens capable ofdetecting antigen-specific antibodies in situ, we have identifiedautoantibodies specific for myelin/oligodendrocyte glycoprotein (MOG)around individual demyelinating axons in acute lesions of both human MSand marmoset EAE, where they appear directly responsible for thedisintegration of myelin sheaths. Animals treated with control F(ab′)₂fragments, i.e., directed against influenza antigen, revealed largedemyelinating plaques in the cervical spinal cord, and animals treatedwith MOG-specific F(ab′)2 fragments showed that the demyelinatingactivity of anti-MOG antibody in marmosets is dependent on intact Fcfragment function, as it can be competitively blocked in vivo byadministration of MOG-specific F(ab′)₂ fragments. These findingsunderscore the role of myelin-specific autoantibodies in the widespreaddestruction of myelin in MS and provide a basis for protective therapyin CNS demyelinating disorders.

Encephalitogenic Epitope Determination (MOG) in MS-like marmoset EAE.

[0065] In C. jacchus marmosets with demyelinating EAE induced withrecombinant rat MOG (rMOG: extracellular domain aa 1-125), the finespecificities of T-cell reactivity (proliferative responses in PBMC) andB-cell reactivity (serum antibody) to MOG were serially studied usingoverlapping 15-mer PIN-peptides (offsets of 1 and 3), corresponding toamino-acid sequences of both rat and human MOG (Chiron Mimotopes, SanDiego, Calif.). Results: All animals studied (n=6) had a prominent andsustained T-cell response restricted to aa 27-36, a sequence totallyconserved across species. A single marmoset responded to a second T-cellepitope located within aa 62-72. Serum antibody responses (n=10) mappedto 4 different regions of MOG including 2 major epitopes, aa 13-21 andaa 62-74 (100% and 60% of animals, respectively) and additional epitopeswere identified in some animals (aa 28-36 and 40-45). No epitopespreading was observed either for T-cells or antibodies in animals withrelapsing EAE that were monitored for up to 93 days. Conclusions:Encephalitogenic responses to MOG in MS-like, marmoset EAE appearrestricted to a limited number of B-cell and T-cell epitopes. Thesefindings demonstrate feasibility of specific immunotherapy in human MS.

Detecting B-cells with surface bound antibodies.

[0066] Early in the immune response B-cells have on their surfaceimmunoglobulins that may specifically react with antigens. The B-cellimmunoglobulin reacting with a self-antigen may be the first step in anautoimmune disease. Thus, early detection of autoantibodies on thesurface of B-lymphocytes may provide the means to design a method oftreatment before the onset of symptoms.

[0067] B-cells constitute about 3-5% of lymphocytes and were positivelyselected from freshly isolated peripheral blood mononuclear cells (PBMC)obtained from C. jacchus marmosets with MOG-induced EAE, from humanswith MS and from healthy controls using anti-CD 19 coated beads.Antibodies to any other suitable B-cell marker may be used. Slidescontaining 2×10⁵ B-cells (>98% purity) were fixed with 1% glutaraldehydeand washed. Alternatively, unfixed cells may be used. The isolatedB-cells were then incubated with labeled immunogold conjugates of amixture of eleven 20-mer overlapping peptides corresponding to thesequence of the NH₂ terminus of human MOG (1-120); identical B-cellpreparations were labeled with control polypeptides corresponding to thesequence of histone or MBP peptides. Slides were enhanced with silverand labeled B-cells were counted by two different blind observers.

[0068] B-cells expressing MOG-specific surface immunoglobulins wereeasily detected with the gold-conjugated MOG peptides in PBMC fromMOG-immunized marmosets (n=8). In these animals which are known todevelop serum anti-MOG antibodies, circulating MOG-specific B-cellsoccurred at a frequency of about 1:500 to about 1:2,000, increased from0-1:10,000 in healthy, unimmunized marmosets (n=8). Unlabeled MOGpeptides added in excess completely inhibited labeling andgold-conjugated control protein failed to label any B-cell. In humans,circulating MOG-specific B-cells could be detected in 8 of 17 MSpatients (47%) and 9 of 18 healthy controls (50%). The frequency ofthese autoreactive B-cells ranged from about 1:11,000 to about 1:200,000B-cells, with the highest frequencies observed in two patients withrelapsing-remitting MS (1:16,000 and 1:11,000, respectively).

[0069] This immunogold assay sensitively detects MOG-specific B-cells inperipheral blood. This assay has a sensitivity of about 1:500,preferably of about 1:2,000, more preferably about 1:10,000, even morepreferably about 1:15,000, and most preferably about 1:200,000. Inhumans, autoreactive B-cells can be detected in approximately 50% ofindividuals, and are equally present in MS patients and controls. Thissuggests that the presence of anti-MOG antibodies in the nervous systemof individuals with MS is not associated with a major expansion ofMOG-reactive B-cells in the peripheral blood. The high frequency ofMOG-reactive B-cells observed in PBMC provides new support for thehypothesis that MOG is an important autoantigen in humans.

[0070] In the Examples above, MOG-specific antibodies were exclusivelylocalized to areas where the transformation of compact myelin into smallvesicles around single demyelinating axons occurred, and to myelindebris either floating in the CNS parenchyma or internalized byphagocytic cells (macrophages and microglia). Thus, in addition to adirect lytic attack on myelin and oligodendrocytes, these antibodies canalso be responsible for receptor-mediated phagocytosis by macrophages,or antibody-dependent cellular cytotoxicity, which have long beenrecognized as possible effector mechanisms of myelin damage. Ourexperiments using passive transfer of antibody in the C. jacchus systemhave shown that it is possible to competitively block pathogenic effectsof the monoclonal anti-MOG antibody 8.18.C5 by in vivo administration ofpurified 8.18.C5-F(ab′)₂ fragments, indicating that intact Fc fragmentsand not the MOG-specific CDR3 sequence themselves mediate the damage tomyelin. Based on these findings, analogs or competitive inhibitors ofantibody binding that are devoid of toxic effects on myelin provide arational approach for therapy in EAE and related demyelinatingdisorders. Thus, the present invention utilizes compositions ofautoantigen epitopes, anti-autoantigen antibody fragments orcombinations thereof to effectuate treatment of demyelinating autoimmunediseases.

[0071] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims. Accordingly, it is intended that the scope of the presentinvention be limited solely by the scope of the following claims,including equivalents thereof.

What is claimed is:
 1. A pharmaceutical composition comprising a MOGpolypeptide-specific antibody fragment sufficient to specifically bind anatural MOG polypeptide and competitively inhibit the binding of anautoantibody to the MOG polypeptide, wherein the fragment does notcomprise a functional Fc portion, and a pharmaceutically acceptablecarrier.
 2. A pharmaceutical composition according to claim 1 furthercomprising a MOG tolerogenic T-cell epitope.
 3. A method for detectingthe presence of an autoantibody bound to a first autoantigen in atissue, comprising the steps of contacting the tissue with a second,labeled autoantigen under conditions wherein the autoantibody binds thesecond autoantigen to form first autoantigen-autoantibody-secondautoantigen labeled complexes, and specifically detecting the labeledcomplexes.
 4. A method according to claim 3, wherein the first andsecond autoantigens are MOG polypeptides.
 5. A method for detecting MOGpolypeptide-specific B-cells comprising the steps of fractionating bloodto obtain an unselected population of B-cells comprising rare MOGpolypeptide-specific B-cells, contacting the population with labeled MOGpolypeptides under conditions whereby the labeled MOG polypeptides bindsthe rare MOG polypeptide-specific B-cells to form labeled complexes ofthe labeled MOG polypeptides and the rare MOG polypeptide-specificB-cells, and specifically detecting the complexes.
 6. A method ofscreening for a candidate agent to inhibit pathology associated with MOGpolypeptide-specific antibody binding to a MOG polypeptide, said methodcomprising the steps of: incubating a mixture comprising: the antibodyor a MOG-specific fragment thereof, the MOG polypeptide, and a candidateagent, under conditions whereby, but for the presence of said agent, theantibody or fragment thereof specifically binds the MOG polypeptide at areference affinity; detecting the binding affinity of antibody orfragment thereof to the MOG polypeptide to determine an agent-biasedaffinity, wherein a diminution of the agent-biased affinity with respectto the reference affinity indicates that said agent inhibits the bindingof the antibody or fragment thereof to the MOG polypeptide and providesa candidate agent for inhibiting pathology associated with MOGpolypeptide-specific antibody binding to a MOG polypeptide.
 7. Apolypeptide comprising a fragment having N and C ends and consisting ofresidues 28-36, 13-21, 67-73, 27-34 or 40-45 of human, rat or marmosetMOG, wherein the fragment is directly joined at at least one of the Nand C-ends with other than natural human or marmoset MOG flankingresidues.
 8. A method of inhibiting MOG-antibody binding comprising thestep of contacting a mixture of a MOG and an antibody with a polypeptideaccording to claim 7, whereby the MOG-antibody binding is inhibited.